本文選題：Nogo66 + 眼用疫苗�。� 參考：《第三軍醫(yī)大學(xué)》2012年碩士論文

【摘要】：原發(fā)性視網(wǎng)膜色素變性（retinitis pigmentosa，RP）是一類(lèi)以感光細(xì)胞和色素上皮層功能障礙為特征的遺傳性視網(wǎng)膜退變疾病， RP以感光細(xì)胞（Photoreceptor cell，PRC）的變性及凋亡為初始病變，隨病程發(fā)展視網(wǎng)膜各層結(jié)構(gòu)、功能均受到嚴(yán)重影響，視網(wǎng)膜二、三級(jí)神經(jīng)元的變性凋亡是RP的重要病理變化。目前臨床上對(duì)RP尚無(wú)特效治療，基因治療、視網(wǎng)膜移植治療、微環(huán)境調(diào)節(jié)（營(yíng)養(yǎng)因子供給、中和或消除毒性及抑制性物質(zhì)）、視覺(jué)假體植入等均在實(shí)驗(yàn)研究中。 視網(wǎng)膜是中樞神經(jīng)系統(tǒng)（Central nervous system，CNS）的一部分，CNS損傷后再生困難一直是神經(jīng)醫(yī)學(xué)研究的難題。Cohen[1]觀察到CNS損傷后在受損神經(jīng)病灶周?chē)写罅縏淋巴細(xì)胞的聚集并出現(xiàn)短暫、微弱的神經(jīng)保護(hù)效應(yīng)，這是因損傷部位自身抗原暴露，刺激T淋巴細(xì)胞活化而產(chǎn)生的保護(hù)性免疫反應(yīng)。根據(jù)這一現(xiàn)象Schwartz等在2000[2]年首次提出：“生理性T細(xì)胞介導(dǎo)的自身免疫性神經(jīng)保護(hù)效應(yīng)”的概念，并于2005年[3]和2009年[4]在Neuroscience中詳細(xì)闡述了用自身抗原髓磷脂相關(guān)蛋白，經(jīng)主動(dòng)免疫和被動(dòng)免疫可誘導(dǎo)并加強(qiáng)這種生理性免疫應(yīng)答的方法，證明生理性免疫應(yīng)答可促進(jìn)神經(jīng)損傷后的再生修復(fù)。其機(jī)制在于通過(guò)啟動(dòng)保護(hù)性自身免疫反應(yīng)抑制損傷神經(jīng)元的繼發(fā)損害，減輕或者防止神經(jīng)損害進(jìn)展，保護(hù)未損傷的神經(jīng)元，幫助修復(fù)處于“損傷邊緣期”的神經(jīng)細(xì)胞。動(dòng)物試驗(yàn)還證實(shí)，上述反應(yīng)缺失時(shí)損傷后果更加嚴(yán)重。 已經(jīng)證實(shí)高眼壓[5]和視神經(jīng)鉗夾傷后[6]視網(wǎng)膜和視神經(jīng)中存在導(dǎo)致神經(jīng)再生修復(fù)困難的內(nèi)源性抑制因子—髓磷脂抑制蛋白，Nogo蛋白是其中之一。有研究指出RP視網(wǎng)膜可表達(dá)Nogo受體Ng-R[7-8]、P75NTR并與視網(wǎng)膜色素變性存在相關(guān)關(guān)系[9][10]。研究髓磷脂抑制因子Nogo是否參與了視網(wǎng)膜色素變性的病理過(guò)程，將為我們利用中樞神經(jīng)系統(tǒng)中存在的生理性自身免疫應(yīng)答機(jī)制，實(shí)現(xiàn)對(duì)RP病理過(guò)程的干預(yù)或者提供神經(jīng)保護(hù)。 中樞神經(jīng)損傷后Nogo釋放增加、表達(dá)增強(qiáng)，發(fā)揮對(duì)損傷神經(jīng)的再生抑制作用，同時(shí)啟動(dòng)神經(jīng)元凋亡過(guò)程，導(dǎo)致神經(jīng)元死亡。Nogo蛋白的碳端和氮端在細(xì)胞膜內(nèi)，而胞膜外由66個(gè)氨基酸殘基形成拓?fù)浣Y(jié)構(gòu)，又稱(chēng)Nogo-66。 Nogo-66通過(guò)與細(xì)胞表面受體Ng-R的配體受體式結(jié)合，介導(dǎo)了Nogo的中樞神經(jīng)抑制活性。本課題組前期利用基因重組技術(shù)[11]，從質(zhì)粒pET-46EK/LIC-Nogo-66原核表達(dá)得到純化的Nogo-66蛋白，分子量7.34Kd，經(jīng)序列分析Nogo-66蛋白存在多處能夠激活T淋巴細(xì)胞的抗原提呈表位肽段，在體外可直接激活視網(wǎng)膜小膠質(zhì)細(xì)胞。并且應(yīng)用重組Nogo66-cs疫苗經(jīng)全身免疫和局部粘膜淋巴免疫，有效的啟動(dòng)了青光眼和視神經(jīng)損傷大鼠的生理性自身免疫反應(yīng)，并證實(shí)有神經(jīng)保護(hù)作用[12]。 皇家外科學(xué)院大鼠（royal college of surgery rat, RCS）是研究RP的經(jīng)典模型，具有和人類(lèi)RP相似的病理變化及功能特征。本實(shí)驗(yàn)以RCS大鼠為研究對(duì)象，首先觀察RCS大鼠自然病程中髓磷脂抑制蛋白Nogo-A/B的表達(dá)情況，然后給予重組Nogo66-cs眼用疫苗進(jìn)行局部粘膜淋巴免疫。通過(guò)檢測(cè)免疫大鼠局部視網(wǎng)膜特異性抗體的表達(dá)研究疫苗的免疫反應(yīng)；觀察比較RCS大鼠自然病程中免疫組和對(duì)照組視網(wǎng)膜的組織病理學(xué)和視網(wǎng)膜神經(jīng)細(xì)胞凋亡情況，研究重組Nogo66-cs眼用疫苗的神經(jīng)保護(hù)作用。同時(shí)檢測(cè)免疫組大鼠和對(duì)照組視網(wǎng)膜中CNTF和bFGF的蛋白表達(dá)情況，探討重組Nogo66-cs眼用疫苗的神經(jīng)保護(hù)機(jī)制。以期進(jìn)一步完善重組Nogo66-cs眼用疫苗滴眼液的免疫策略，拓展其使用范圍。 一、主要研究?jī)?nèi)容 1.髓磷脂抑制蛋白Nogo-A/B在RCS視網(wǎng)膜自然病程中的表達(dá)取出生后15d、30d、60d和90d四個(gè)時(shí)間點(diǎn)的視網(wǎng)膜含色素變性大鼠（RCS-p+)各5只為實(shí)驗(yàn)組，相同時(shí)間點(diǎn)視網(wǎng)膜含色素正常大鼠（RCS-rdy+p+)各5只為正常對(duì)照組。利用免疫組化和免疫印跡分別定性和定量檢測(cè)視網(wǎng)膜中Nogo-A/B的表達(dá)。實(shí)驗(yàn)動(dòng)物由第三軍醫(yī)大學(xué)大坪醫(yī)院野戰(zhàn)外科研究所實(shí)驗(yàn)動(dòng)物中心提供。 2.重組Nogo66-cs眼用疫苗經(jīng)粘膜淋巴免疫誘導(dǎo)保護(hù)性免疫應(yīng)答效應(yīng)和方式取出生后20天RCS-p+大鼠為研究對(duì)象，，隨機(jī)分為Nogo66-cs疫苗組和CS組，各9只。免疫接種分3組：第一組，初次免疫后追加免疫1次（1次/周），末次免疫后7d取材；第二組，初次免疫后追加免疫2次（1次/周），末次免疫后7d取材；第三組，初次免疫后追加免疫3次（1次/周），末次免疫后7d取材；對(duì)照組以相同策略給予CS點(diǎn)眼。采用TUNEL原位末端凋亡法檢測(cè)視網(wǎng)膜凋亡陽(yáng)性表達(dá)，IPP分析視網(wǎng)膜厚度變化，以比較追加免疫對(duì)視網(wǎng)膜結(jié)構(gòu)的影響。采用免疫印跡檢測(cè)視網(wǎng)膜IgG表達(dá)，以明確重組Nogo66-cs眼用疫苗是否可經(jīng)粘膜淋巴免疫誘導(dǎo)視網(wǎng)膜局部免疫反應(yīng)。 3.重組Nogo66-cs眼用疫苗對(duì)RCS視網(wǎng)膜免疫神經(jīng)保護(hù)作用研究取出生后30天RCS-p+為實(shí)驗(yàn)對(duì)象，隨機(jī)分為重組Nogo66-cs眼用疫苗和CS組，各5只。雌雄不限。實(shí)驗(yàn)組初次免疫后追加免疫2次（1次/周），末次免疫后7d取材，對(duì)照組采取相同策略。采用IPP檢測(cè)視網(wǎng)膜厚度及TUNEL凋亡陽(yáng)性表達(dá)；探討重組Nogo66-cs眼用疫苗對(duì)RP視網(wǎng)膜細(xì)胞的免疫神經(jīng)保護(hù)作用。 4.重組Nogo66-cs眼用蛋白疫苗對(duì)RCS大鼠視網(wǎng)膜的免疫性神經(jīng)保護(hù)機(jī)制研究實(shí)驗(yàn)分組及免疫策略同前。采用免疫組化法、免疫印跡法檢測(cè)兩組視網(wǎng)膜睫狀神經(jīng)生長(zhǎng)因子（Ciliary neurotrophic factor，CNTF）、堿性成纖維生長(zhǎng)因子（Basic fibroblast growthfactor，bFGF）蛋白表達(dá)。探討重組Nogo66-cs眼用疫苗對(duì)RP的免疫性神經(jīng)保護(hù)機(jī)制。 二、主要結(jié)果 （一）Nogo-A/B在RCS大鼠視網(wǎng)膜中的表達(dá) 1.組織病理學(xué)改變與對(duì)照組相比，P15d和P30d，RCS-p+大鼠視網(wǎng)膜外層細(xì)胞結(jié)構(gòu)和數(shù)目未出現(xiàn)明顯變化，P30d內(nèi)核層(Inner nuclear layer，INL)層細(xì)胞排列出現(xiàn)輕微紊亂；P60d、P90d大鼠視網(wǎng)膜外核層(Outer nuclear layer，ONL)和INL結(jié)構(gòu)出現(xiàn)明顯紊亂，細(xì)胞數(shù)目減少以O(shè)NL和神經(jīng)節(jié)細(xì)胞層(Ganglion cell layer，RGC)層較為明顯。提示RCS大鼠視網(wǎng)膜色素變性發(fā)展過(guò)程中，視網(wǎng)膜外層和視網(wǎng)膜內(nèi)層結(jié)構(gòu)均出現(xiàn)改變。 2. Nogo-A/B的免疫組化和免疫印跡顯示Nogo-A/B蛋白在RCS-p+實(shí)驗(yàn)大鼠各時(shí)間段視網(wǎng)膜表達(dá)均為陽(yáng)性，主要位于視網(wǎng)膜INL和RGC層。Nogo-A/B蛋白在RCS-rdy+p+對(duì)照大鼠各時(shí)間段視網(wǎng)膜表達(dá)為弱陽(yáng)性表達(dá)。 WB免疫印記檢測(cè)RCS-P+大鼠P15d、P30d、P60d和P90d Nogo-A蛋白表達(dá)量分別為：0.82737±0.21292、1.11019±0.08999、1.31552±0.02857、1.26881±0.08042，組間比較存在顯著差異(P＜0.05)，各時(shí)間點(diǎn)組內(nèi)比較存在統(tǒng)計(jì)學(xué)差異（P＜0.05）。提示RCS大鼠的RP變性過(guò)程中存在內(nèi)源性髓磷脂抑制蛋白Nogo-A/B的動(dòng)態(tài)表達(dá)變化，表明Nogo-A蛋白參與RP病理過(guò)程。 （二）追加免疫后視網(wǎng)膜結(jié)構(gòu)變化和局部免疫效應(yīng) 1.追加免疫效應(yīng)分析初次免疫后追加免疫1次、2次和3次，與對(duì)照組相比，實(shí)驗(yàn)組視網(wǎng)膜INL厚度分別為：12.4581±2.64716（P＜0.05）、11.0671±2.38886（P＜0.05）、8.94238±0.82968（P＞0.05）。與對(duì)照組相比，實(shí)驗(yàn)組視網(wǎng)膜ONL厚度分別為： 11.6328±1.77681（P＜0.05）、15.4117±4.66376（P＜0.05）、11.3383±4.61539（P＞0.05）。 TUNEL分析視網(wǎng)膜凋亡陽(yáng)性表達(dá)，初次免疫后追加免疫1、2、3次，實(shí)驗(yàn)組單位面積內(nèi)凋亡陽(yáng)性表達(dá)IOD sum/Area比值分別為：0.060365219±0.060365（P＞0.01）、0.03565282±0.019462（P＜0.01）、0.107844636±0.107845（P＞0.01）。與對(duì)照組相比差異具有統(tǒng)計(jì)學(xué)意義。提示2次追加免疫可延緩RP所致視網(wǎng)膜厚度變薄和視網(wǎng)膜細(xì)胞的進(jìn)行性凋亡。 2.視網(wǎng)膜中IgG抗體檢測(cè)結(jié)果經(jīng)重組Nogo66-cs眼用疫苗2次追加免疫RCS大鼠， WB檢測(cè)實(shí)驗(yàn)、對(duì)照組視網(wǎng)膜IgG抗體表達(dá)量分別為：1.15435±0.25090、0.43957±0.13643，兩組比較差異具有顯著統(tǒng)計(jì)學(xué)意義（P＜0.01）。提示重組Nogo66-cs眼用疫苗可經(jīng)粘膜淋巴免疫誘導(dǎo)視網(wǎng)膜局部特異性免疫反應(yīng)。 （三）重組Nogo66-cs眼用疫苗免疫后視網(wǎng)膜TUNEL和厚度變化 1.視網(wǎng)膜凋亡細(xì)胞計(jì)數(shù)IPP半定量分析視網(wǎng)膜TUNLEL凋亡陽(yáng)性表達(dá)，與對(duì)照組相比，實(shí)驗(yàn)組IOD SUM/Area為：0.0576±0.0038（P＜0.05），兩組間比較差異具有統(tǒng)計(jì)學(xué)意義 2.視網(wǎng)膜厚度IPP分析視網(wǎng)膜各層厚度，與對(duì)照組相比，實(shí)驗(yàn)組INL層厚度為：13.4905±0.6211（P＜0.01）；ONL層厚度為：4.8293±0.5943（P＜0.05）。表明重組Nogo66-cs眼用疫苗免疫接種后可有效抑制RP所致視網(wǎng)膜神經(jīng)細(xì)胞進(jìn)行性凋亡，并延緩視網(wǎng)膜各層厚度變薄，以INL明顯。 （四）CNTF和bFGF免疫組化和免疫印跡 1.免疫組化重組Nogo66-cs眼用疫苗免疫接種RCS大鼠后可誘導(dǎo)CNTF和bFGF在RCS視網(wǎng)膜上的陽(yáng)性表達(dá)，CNTF表達(dá)以INL和RGC層為主，bFGF主要表達(dá)在RGC層； 2.免疫印跡WB檢測(cè)實(shí)驗(yàn)、對(duì)照組視網(wǎng)膜bFGF表達(dá)分別為：0.82572±0.02803、0.60233±0.04789，組間比較差異具有顯著統(tǒng)計(jì)學(xué)意義（P＜0.01）；WB檢測(cè)實(shí)驗(yàn)、對(duì)照組視網(wǎng)膜CNTF表達(dá)分別為：0.91272±0.19833、0.60759±0.09207，組間比較差異具有統(tǒng)計(jì)學(xué)意義（P＜0.05）；bFGF表達(dá)兩組比較差異相對(duì)較高。研究表明重組Nogo66-cs眼用疫苗延緩RP視網(wǎng)膜變性的機(jī)制之一是內(nèi)源性bFGF、CNTF的營(yíng)養(yǎng)支持。 結(jié)論： 1. Nogo-A蛋白參與RP變性過(guò)程。 2.重組Nogo66-cs眼用疫苗經(jīng)粘膜淋巴免疫可有效誘導(dǎo)視網(wǎng)膜局部特異性免疫反應(yīng)。 3.重組Nogo66-cs眼用疫苗對(duì)RP視網(wǎng)膜具有免疫性神經(jīng)保護(hù)效應(yīng)，其機(jī)制之一是內(nèi)源性bFGF、CNTF的營(yíng)養(yǎng)支持。
[Abstract]:Retinitis pigmentosa (RP) is a kind of hereditary retinal degeneration disease characterized by the dysfunction of photosensitive cells and pigment epithelium. RP is the initial lesion of the degeneration and apoptosis of the Photoreceptor cell (PRC), and the structure of the retina is developed with the course of the disease. The function of the retina is seriously affected. The degeneration and apoptosis of the two and three stage neurons of the membrane is an important pathological change of RP. There is no special treatment for RP at present. Gene therapy, retina transplantation, microenvironmental regulation (nutrient supply, neutralization or elimination of toxic and inhibitory substances), and visual prosthesis implantation are all in the experimental study.
The retina is part of the central nervous system (Central nervous system, CNS). The difficulty of regeneration after CNS injury has been a difficult problem in neurology..Cohen[1] observed the accumulation of a large number of T lymphocytes around the damaged nerve lesion after CNS injury and a transient, weak neuroprotective effect, which is due to the self antigen storm of the injured site. A protective immune response to the activation of T lymphocytes. According to this phenomenon, the concept of "physiological T cells mediated autoimmune neuroprotective effects" was first proposed in 2000[2], according to this phenomenon, and in [3] and [4] in 2009, in 2005, the myelin related protein of the autoantigen was explained in Neuroscience, and the main body of the protein was expressed in Neuroscience. Dynamic and passive immunity can induce and strengthen this physiological response. It is proved that the physiological immune response can promote regeneration and repair after nerve injury. The mechanism is to inhibit the secondary damage of injured neurons by activating the protective autoimmune reaction, reduce or prevent the progression of nerve damage, and protect the undamaged nerves. It also helped to repair nerve cells at the "edge of injury". Animal tests also confirmed that the loss of these reactions was more serious.
The endogenous inhibitory factor, myelin suppressor protein, which has been found in the [6] retina and optic nerve, is one of the endogenous inhibitors of myelin suppressor, which has been found in the retina and optic nerve of the retina and optic nerve after [5] and optic nerve clamp injury. There is a study that the RP retina can express the Nogo receptor Ng-R[7-8], P75NTR and the relationship with retinal pigment degeneration [9][10]. The study of whether the myelin suppressor factor (Nogo) is involved in the pathological process of retinitis pigmentosa will provide us with the mechanism of physiological autoimmune response in the central nervous system to intervene in the pathological process of RP or to provide neuroprotection.
After the central nerve injury, the release of Nogo increases, the expression is enhanced, and the regeneration of the injured nerve is inhibited, and the process of neuronal apoptosis is initiated, which leads to the neuronal death of the carbon end and nitrogen end of the.Nogo protein in the cell membrane, and the extracellular matrix is formed by 66 amino acid residues, which is also called Nogo-66. Nogo-66 through the cell surface receptor Ng. The ligand receptor binding of -R mediates the central nervous inhibitory activity of Nogo. In our group, we used gene recombination technology [11] to express the purified Nogo-66 protein and molecular weight 7.34Kd from the plasmid pET-46EK/LIC-Nogo-66 prokaryotic. The sequence analysis of Nogo-66 protein has several antigen presenting epitopes capable of activating the T lymphocyte. In vitro, the retinal microglia can be activated directly. And the recombinant Nogo66-cs vaccine is used to activate the physiological autoimmune reaction of the rats with glaucoma and optic nerve injury through systemic and local mucosal lymphoid immunization, and the neuroprotective effect of [12]. is confirmed.
The Royal surgery College (Royal College of surgery rat, RCS) is the classic model of the study of RP, with the pathological changes and functional features similar to human RP. The experiment was conducted to observe the expression of the Nogo-A/B of myelin suppressor protein in the natural course of RCS rats, and then the recombinant Nogo66-cs eye vaccine was given. Local mucosal lymphoid immunity was carried out. The immunoreaction of the vaccine was studied by detecting the expression of local retina specific antibody in the immune rats. The histopathology of the retina and the retinal nerve cell apoptosis in the immune and control groups of the RCS rats were observed and compared. The neuroprotective effect of the recombinant Nogo66-cs eye vaccine was studied. At the same time, the protein expression of CNTF and bFGF in the retina of the immune group and the control group was detected, and the neuroprotective mechanism of the recombinant Nogo66-cs eye vaccine was explored in order to further improve the immunization strategy of the recombinant Nogo66-cs eye drops and expand its scope of use.
First, the main research content
1. the expression of myelin suppressor protein Nogo-A/B in the natural course of RCS retina takes 5 retinal pigment degeneration rats (RCS-p+) at the four time points of 15d, 30d, 60d and 90d, each of the experimental group, and 5 of the retinal pigment normal rats (RCS-rdy+p+) at the same time point as the normal control group. Using immunohistochemistry and immunoblotting, respectively. The expression of Nogo-A/B in the retina was qualitatively and quantitatively examined. The experimental animals were provided by the experimental animal center of the Field Surgery Institute of Daping Hospital, Third Military Medical University.
2. the RCS-p+ rats of the recombinant Nogo66-cs eye vaccine were divided into Nogo66-cs vaccine group and CS group, each of which were randomly divided into 3 groups: the first group, the first group, 1 times after the first immunization (1 times / week), and the 7d after the last immunization; the second group, the first group. After immunization, 2 times (1 times / week) and 7d were taken after the last immunization; the third groups were immunized with 3 times (1 times / weeks) after the first immunization, 7d was obtained after the last immunization, and the control group was given CS points with the same strategy. The positive expression of retinal apoptosis was detected by TUNEL in situ end apoptosis method, and IPP was used to analyze the retinal thickness changes in order to compare additional immunity. The effects of pestilence on retinal structure were detected by immunoblotting to detect the expression of retina IgG in order to determine whether the recombinant Nogo66-cs eye vaccine could induce local retinal immune response through mucosal lymphatic immunization.
3. the study of the protective effect of recombinant Nogo66-cs eye vaccine on RCS retina immuno neuroprotection took RCS-p+ 30 days after birth as the experimental object, randomly divided into the recombinant Nogo66-cs eye vaccine and the CS group, each 5. The experimental group was immunized with 2 times (1 times / week) after the first immunization, the same strategy was taken after the last immunization, and the control group adopted the same strategy. IPP examination was adopted. Retinal thickness and positive expression of TUNEL apoptosis were measured. The immuno neuroprotective effect of recombinant Nogo66-cs ophthalmic vaccine on RP retinal cells was investigated.
4. the experimental group and immunization strategy of recombinant Nogo66-cs eye protein vaccine on RCS rat retina were divided into two groups: immunohistochemistry and Western blotting to detect the two groups of Ciliary neurotrophic factor (CNTF) and basic fibroblast growth factor (Basic fibroblast growthfacto). R (bFGF) protein expression. To explore the immune neuroprotective mechanism of recombinant Nogo66-cs ophthalmic vaccine on RP.
Two, the main results
(1) expression of Nogo-A/B in the retina of RCS rats
1. compared with the control group, there was no obvious change in the structure and number of cells in the outer retina of P15d and P30d rats, and a slight disorder in the P30d kernel layer (Inner nuclear layer, INL) layer cells, and there were obvious disorders in the outer retinal nucleus (Outer nuclear) and the structure of the P90d rat, and the number of cells. The decrease of the layer of ONL and the ganglion cell layer (Ganglion cell layer, RGC) is more obvious. It is suggested that the outer layer of retina and the inner layer of the retina change in the development of retinal pigment degeneration in RCS rats.
2. Nogo-A/B immunoblotting and immunoblotting showed that the expression of Nogo-A/B protein was positive in all time segments of RCS-p+ experimental rats, mainly located in the retina INL and RGC layer.Nogo-A/B protein in the RCS-rdy+p+ control rats, and the expression of retina was weak positive. WB immunoimprint was used to detect P15d, P30d, P60d and the.Nogo-A/B in RCS-P+ rats. The expression of o-A protein was 0.82737 + 0.21292,1.11019 + 0.08999,1.31552 + 0.02857,1.26881 + 0.08042, and there was a significant difference between the groups (P < 0.05), and there was a statistical difference in each time point group (P < 0.05). It suggested that the dynamic expression of the endogenous myelin suppressor protein Nogo-A/B in the RP denaturation process of RCS rats The Nogo-A protein is involved in the pathological process of RP.
(two) retinal structural changes and local immune effects after supplemental immunization
1. the additional immunization effect was analyzed for 1 times, 2 times and 3 times after primary immunization. Compared with the control group, the retinal INL thickness was 12.4581 + 2.64716 (P < 0.05), 11.0671 + 2.38886 (P < 0.05), 8.94238 + 0.82968 (P > 0.05). Compared with the control group, the retinal ONL thickness was respectively:
11.6328 + 1.77681 (P < 0.05), 15.4117 + 4.66376 (P < 0.05), 11.3383 + 4.61539 (P > 0.05).
TUNEL analyzed the positive expression of retinal apoptosis and added immunization 1,2,3 times after the first immunization. The positive expression of IOD sum/Area in the experimental group was 0.060365219 + 0.060365 (P > 0.01), 0.03565282 + 0.019462 (P < 0.01), 0.107844636 + 0.107845 (P > 0.01). The difference was statistically significant compared with the control group. 2 times. Supplemental immunization can delay retinal thinning and progressive apoptosis of retinal cells induced by RP.
2. the results of IgG antibody detection in the retina were added to the recombinant Nogo66-cs eye vaccine for 2 additional immunization of RCS rats and WB test. The expression of IgG antibody in the retina of the control group was 1.15435 + 0.25090,0.43957 + 0.13643 respectively. The two groups had significant statistical significance (P < 0.01). The pestilence induced local specific immunoreaction of the retina.
(three) changes in TUNEL and thickness of retina after immunization with recombinant Nogo66-cs ophthalmic vaccine.
1. retinal apoptotic cells count IPP semi quantitative analysis of retinal TUNLEL apoptosis positive expression, compared with the control group, IOD SUM/Area in the experimental group was 0.0576 + 0.0038 (P < 0.05). The difference of the two groups was statistically significant.
2. retinal thickness IPP was used to analyze the thickness of each layer of the retina. Compared with the control group, the thickness of the INL layer in the experimental group was 13.4905 + 0.6211 (P < 0.01), and the thickness of the ONL layer was 4.8293 + 0.5943 (P < 0.05). It indicated that the recombinant Nogo66-cs eye vaccine could effectively inhibit the sexual apoptosis of the retinal membrane neurons caused by RP and postpone the retina layers after the immunization of the recombinant Nogo66-cs eye. The thickness is thinner, with INL obvious.
(four) immunoblotting and immunoblotting of CNTF and bFGF
1. immunized recombinant Nogo66-cs eye vaccine inoculated RCS rats could induce positive expression of CNTF and bFGF on RCS retina. The expression of CNTF was mainly in INL and RGC layer, and bFGF was mainly expressed in the RGC layer.
2. immunoblotting WB test showed that the expression of bFGF in the retina of the control group was 0.82572 + 0.02803,0.60233 + 0.04789, and the difference between the groups was statistically significant (P < 0.01). The CNTF expression in the retina of the control group was 0.91272 + 0.19833,0.60759 + 0.09207 respectively, and the difference between the groups was statistically significant (P < 0.05). The comparison of bFGF expression between two groups was relatively high. The study showed that one of the mechanisms of recombinant Nogo66-cs eye vaccine to delay RP retinal degeneration was endogenous bFGF and CNTF nutritional support.
Conclusion:
1. Nogo-A protein participates in the RP denaturation process.
2. recombinant Nogo66-cs ophthalmic vaccine can effectively induce the local specific immune response of the retina through mucosal lymphatic immunity.
3. recombinant Nogo66-cs ophthalmic vaccine has an immunological neuroprotective effect on RP retina, one of which is endogenous bFGF and CNTF nutritional support.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R774.1

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